Design, synthesis, and biological evaluations of novel 7-[7-amino-7-methyl- 5-azaspiro[2.4]heptan-5-yl]-8-methoxyquinolines with potent antibacterial activity against respiratory pathogens(1)

Article abstract of DOI:10.1021/jm301650g

Novel 7-[7-amino-7-methyl-5-azaspiro[2.4]heptan-5-yl]-6-fluoro-1-[(1R,2S)- 2-fluorocyclopropyl]- 8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylic acid 2a and 2b were designed and synthesized to obtain potent antibacterial drugs for the treatment of respi

Full text of DOI:10.1021/jm301650g

Article
pubs.acs.org/jmc
Design, Synthesis, and Biological Evaluations of Novel 7‑[7-Amino-7-
methyl-5-azaspiro[2.4]heptan-5-yl]-8-methoxyquinolines with Potent
Antibacterial Activity against Respiratory Pathogens1
Takashi Odagiri,* Hiroaki Inagaki, Yuichi Sugimoto, Masatoshi Nagamochi, Rie N. Miyauchi,
Junichi Kuroyanagi, Takahiro Kitamura, Satoshi Komoriya, and Hisashi Takahashi
R&D Division, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
S
* Supporting Information
ABSTRACT: Novel 7-[7-amino-7-methyl-5-azaspiro[2.4]heptan-5-yl]-6-fluoro-1-[(1R,2S)-2-fluo-
rocyclopropyl]- 8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylic acid 2a and 2b were designed
and synthesized to obtain potent antibacterial drugs for the treatment of respiratory tract infections.
Among these, compound 2a possessing (S)-configuration for the asymmetrical carbon on the
pyrolidine moiety at the C-7 position of the quinolone scaffold exhibited potent in vitro
antibacterial activity against respiratory pathogens including Gram-positive (Streptococcus
pneumoniae and Staphylococcus aureus), Gram-negative (Haemophilus influenzae and Moraxcella
catarrhalis), and atypical strains (Chalmydia pneumoniae and Mycoplasma pneumoniae), as well as
multidrug-resistant Streptococcus pneumoniae and quinolone-resistant and methicillin-resistant
Staphylococcus aureus). Furthermore, compound 2a showed excellent in vivo activity against the experimental murine pneumonia
model due to multidrug resistant Streptococcus pneumoniae (MDRSP) and favorable profiles in preliminary toxicological and
nonclinical pharmacokinetic studies.
great number of medicines as substrates. Although beneficial
combination therapy utilizing CYP3A4 inhibition has been
reported, clinical drug−drug interaction (DDI) due to CYP3A4
inhibition often resulted in serious adverse events.9,10 There-
fore, we judged compound 1 to be inappropriate as a new
candidate for further development.
INTRODUCTION
■
As community-acquired pathogens are exhibiting increasing
levels of resistance to β-lactams and macrolides, newer
quinolones have emerged as one of the first-line antibacterial
drugs for respiratory tract infections in the medical community.
Meanwhile, multidrug-resistant Streptococcus pneumoniae
(MDRSP) and community-acquired methicillin-resistant Staph-
ylococcus aureus (CA-MRSA) are emerging as key pathogens in
community-acquired respiratory infections.2,3 Fluoroquinolones
such as levofloxacin (LVFX), gatifloxacin (GFLX), and
moxifloxacin (MFLX) are beneficial for the empirical treatment
of respiratory infections in the community because of their
extended antibacterial spectra, including atypical pathogens,
with preferable pharmacokinetic (PK) and safety profiles;4
however, the antibacterial activity of these newer quinolones
are not potent enough, and bacteria resistance such as
quinolone-resistant S. pneumoniae and CA-MRSA to these
drugs will be problematic in the near future.5 On the other
hand, clinical adverse events (e.g., torsades de pointes, or fatal
liver injury) by some quinolones have been reported.6,7 Thus,
the new respiratory quinolone providing both improved activity
against respiratory pathogens including MDRSP and a good
safety profile is keenly required.
To solve the MBI problem, we focused on the chemical
structure at the C-7 pyrrolidine substituent of compound 1.
According to the report by Silverman,11 the chemical structure
like the cyclopropylmethylamine unit may have a risk to
inactivate the enzyme monoamine oxidase (MAO) and the
inactivation begins with the formation of iminium intermediate.
That is to say, the conversion to the structure that cannot form
an iminium intermediate may prevent the exertion of MBI. On
the other hand, trovafloxacin is reported to show numerous
cases of hepatotoxicity, which limits its clinical usefulness. It
possesses two substructural elements that have the potential to
generate reactive intermediates: a cyclopropylamine moiety and
a difluoroanilino system. Evidence has been reported that
trovafloxacin-induced hepatotoxicity may be mediated through
oxidation of the cyclopropylamine substructure to reactive
intermediates that may form covalent adducts to hepatic
proteins, resulting in damage to liver tissue.12 Therefore, we
planned to introduce a methyl group onto the carbon atom at
the root of the C7-amino moiety of compound 1, thereby
aiming to avoid the formation of the reactive intermediate.
As previously reported,8 compound 1 showed potent activity
against respiratory pathogens including penicillin-resistant S.
pneumoniae (PRSP), together with a superior safety profile
similar to levofloxacin (LVFX). However, the results of the in
vitro metabolic study revealed that compound 1 exerted
mechanism-based inhibition (MBI) of cytochrome (CYP) 3A4.
CYP3A4 is the most abundant CYP isozyme that accepts a
Received: November 8, 2012
Published: February 14, 2013
© 2013 American Chemical Society
1974
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Article
Figure 1.
Scheme 1
a
Scheme 2
a
Reagents: (a) NaCN, NH3 aq, NH4Cl, NH3/MeOH; (b) H2, Raney Ni/EtOH; (c) concentrated aqueous HCl; (d) 1,1,1,3,3,3-
hexamethyldisilazane/MeCN; (e) Boc2O/1.4-dioxane; (f) BnBr, NaH/DMF; (g) Chiralpak AD; (h) TFA/CH2Cl2; (i) LiAlH4/THF; (j)
Boc2O/CH2Cl2; (k) H2, Pd−C/MeOH; (l) 4, Et3N/DMSO; (m) Et3N/80% aqueous EtOH; (n) concentrated aqueous HCl.
In this article, we describe the details of the synthesis, in vitro
and in vivo antibacterial activities, safety profile, and
pharmacokinetic profile of the designed compound 2a (Figure
1).
acidic condition gave diaminocarboxylic acid DL-7. Whne DL-7
was heated with 1,1,1,3,3,3-hexamethyldisilazane in acetonitrile
(MeCN), the desired cyclization was accomplished. The
remaining amino moiety was protected by di-tert-butyl
dicarbonate (Boc2O) to provide lactam DL-8. Treatment of
DL-8 with sodium hydride (NaH) in N,N-dimethylformamide
(DMF) followed by a reaction with benzyl bromide (BnBr)
provided benzylamide DL-9. This racemic 9 was separated into
the two enantiomers (+)-9 and (−)-9 by column chromatog-
raphy with Chiralpak AD. After the temporary removal of tert-
butoxycarbonyl groups of (+)-9 and (−)-9, the amide moieties
were reduced by lithium aluminum hydride (LiAlH4). Then the
primary amino groups were protected again by Boc2O to give
benzylamines (+)-10 and (−)-10. The benzyl groups were
CHEMISTRY
■
As shown in Scheme 1, we envisioned synthesizing the desired
7-substituted 8-methoxyquinolone derivative 2a by aromatic
nucleophilic substitution reaction from amine 3a and 6,7-
difluoro-1-[(1R,2S)-2-fluorocyclopropan-1-yl]-8-methoxy-4-
oxo-1,4-dihydroquinoline-3-carboxylic acid BF2 chelate (4).13
The synthesis of 2 is illustrated in Scheme 2. Amine DL-6 was
prepared by the Strecker reaction of 5.14 Hydrogenation with
Raney Ni and the removal of the tert-butyl group under an
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Journal of Medicinal Chemistry
Article
a
Scheme 3
a
Reagents: (a) LiHMDS/THF; (b) TBAF/THF; (c) PhSO2Cl, Et3N, DMAP/CH2Cl2; (d) NaHMDS/THF; (e) TFA/CH2Cl2; (f)
diphenylphosphoryl azide, Et3N/toluene; (g) 6 N aqueous HCl/1,4-dioxane; (h) sodium bis(2-methoxyethoxy)aluminum hydride/toluene; (i)
Boc2O/toluene; (j) H2, Pd−C/MeOH; (k) 4, Et3N/DMSO; (l) Et3N/80% aqueous EtOH; (m) concentrated aqueous HCl.
removed by catalytic hydrogenolysis. The resultant crudes and
quinolonecarboxylic acid BF2 chelate 4 were heated with
triethylamine in dimethyl sulfoxide (DMSO), followed by
dechelation and deprotection to give compounds 2a and 2b.
The absolute configurations of 2a and 2b were determined
by an alternative synthesis (Scheme 3). Alkylation of
compound 11,15 which was synthesized from (R)-phenylethyl-
amine, with use of lithium hexamethyldisilazide (LHMDS),
followed by the treatment with tetrabutylammonium fluoride
(TBAF) provided alcohol 13 as a diastereomixture. After the
conversion of the primary alcohol of 13 to the benzenesulfo-
nyloxy group, the cyclization was performed by treatment with
sodium hexamethyldisilazide (NaHMDS). Compound 14 was
obtained as prisms, and the absolute configuration was
determined to be 7S by X-ray crystallographic analysis (Figure
2). Phenylethylamine 16 was prepared by multistep reactions
(removal of tert-butyl ester, Curtius rearrangement, acidic
hydrolysis, reduction with sodium bis(2-methoxyethoxy)-
aluminum hydride, and formation of tert-butyl carbamate).
After the phenylethyl group of 16 was removed by catalytic
hydrogenolysis, the resultant secondary amine was reacted with
BF2 chelate 4 followed by the above-mentioned method to give
the target compound 2. The analytical data of 2 corresponded
with those of 2a.
RESULTS AND DISCUSSION
■
Figure 2.
The minimum inhibitory concentrations (MICs) of 2a and 2b
against several representative Gram-positive and Gram-negative
bacteria are summarized in Table 1, along with the data for
levofloxacin, moxifloxacin, ciprofloxacin, and the previously
reported compound 1 for comparison. The synthesized
compounds 2a and 2b exhibited a broad antibacterial spectrum
against Gram-positive and Gram-negative bacteria. A novel
quinolone 2a, which has the (7S)-amino group of the C-7
substituent, exhibited about 4 to >16 times better activity
against Gram-positive bacteria compared with the activity of
LVFX, although activities of 2a against some bacteria were
slightly inferior to those of 1. Particularly, 2a was more potent
than an existing respiratory quinolone, moxifloxacin, against
MDRSP and levofloxacin-resistant MRSA strains. Against
Gram-negative bacteria except Pseudomonas aeruginosa, the
activity of 2a was comparable to those of other quinolones
tested. For example, 2a showed potent activities against
Haemophilus influenzae and Moraxcella catarrhalis.
Next, the activity of 2a and representative respiratory
quinolones against atypical organisms are listed in Table 2. A
newer quinolone 2a exhibited potent antibacterial activity
against Mycoplasma pneumoniae and Chlamydophilia pneumo-
niae strains. The activity of 2a was comparable with those of
moxifloxacin and gemifloxacin and was 4−10 times higher than
that of levofloxacin.
Finally, the MICs against clinically isolated levofloxacin-
intermediate and -resistant Streptococcus pneumoniae of
compound 2a and reference antibacterial agents are shown in
Table 3. Compound 2a showed more potent activity than the
other quinolone antibacterial agents (levofloxacin, ciproflox-
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Table 1. Antibacterial Activities MIC (μg/mL) of Compounds 2a and 2b and Reference Quinolones against Gram-Positive and
a
Gram-Negative Bacteria
compd
organism
2a
2b
LVFX
CPFX
MFLX
1
S. aureus FDA 209-P
0.025
0.39
0.1
0.05
1.56
0.2
0.1
>6.25
0.39
0.1
0.05
0.78
0.1
0.012
0.39
0.05
0.05
0.20
0.05
0.1
b
S. aureus 870307
>6.25
0.2
S. epidermidis 56500
c
S. pneumoniae J24
0.05
0.39
0.2
0.39
0.78
0.78
0.1
d
S. pneumoniae 104835
S. pyogenes G-36
>6.25
0.78
>6.25
1.56
3.13
0.2
0.78
0.78
0.39
0.025
0.1
E. faecalis ATCC 19433
B. subtilis IID 685
0.2
0.78
0.78
0.2
0.1
1.56
3.12
0.2
0.05
0.006
0.05
<0.003
0.012
0.2
E. coli NIHJ
0.012
0.05
0.012
0.05
0.78
0.012
0.05
<0.003
0.025
0.006
0.025
0.05
0.012
0.1
K. pneumoniae TYPE 1
H. influenzae ATCC49247
M(B). catarrhalis ATCC25238
P. aeruginosa PAO-1
0.006
0.05
0.78
0.012
0.025
0.39
0.012
0.05
0.78
a
Antibacterial activities were determined using a standard microbroth dilution method. Abbreviations are as follows: LVFX, levofloxacin; CPFX,
b
c
ciprofloxacin; MFLX, moxifloxacin. Levofloxacin-resistant and methicillin-resistant S. aureus (levofloxacin-r-MRSA). Penicillin-susceptible S.
pneumoniae (PSSP). Multidrug-resistant S. pneumoniae (MDRSP, quinolone-resistant and penicillin-resistant strains).
d
Table 2. Antibacterial Activities MIC (μg/mL) of 2a and Reference Quinolones against Atypical Organisms Mycoplasma
a
pneumoniae and Chlamydophilia pneumoniae
compd
organism
2a
LVFX
MFLX
GRNX
GMFX
M. pneumoniae 18-1
0.12
2
0.25
0.12
0.25
M. pneumoniae ATCC 29085
C. pneumoniae AR-39
C. pneumoniae CM-1
0.12
1
0.12
0.06
0.25
0.125
0.063
0.5
0.5
0.125
0.125
0.063
0.016
0.125
0.063
a
Antibacterial activities of M. pneumoniae and C. pneumoniae strains were determined using a standard microbroth dilution method and standard
recommended techniques, respectively. Abbreviations are as follows: LVFX, levofloxacin; MFLX, moxifloxacin; GRNX, garenoxacin; GMFX,
gemifloxacin.
generally in the clinic as antibacterials for the treatment of
respiratory tract infections. The MIC90 of 2a was 1 μg/mL,
equal to that of garenoxacin. Thus, 2a exhibited potent and well
balanced antibacterial activity against representative respiratory
pathogens, including atypical strains and MDRSP.
Table 3. Antibacterial Activities MIC (μg/mL) of 2a and
Reference Drugs against Clinical Isolates of Levofloxacin-
Intermediate and -Resistant Streptococcus pneumoniae
a
b
MIC (μg/mL)
compd
range
MIC50
MIC80
MIC90
The potential inhibitory effects of compounds 1 and 2a on a
mechanism-based inhibition (MBI) of CYP3A4 were inves-
tigated using the general procedure described in the
Experimental Section. The results are illustrated in Figures 3
and 4. Compound 1 inhibited the activity of CYP3A4 for 1′-
hydroxylation of midazolam dose-dependently; however, 2a did
not show an inhibitory effect on the MBI of CYP3A4 over the
range of concentrations tested. According to these experimental
findings, introduction of the methyl group into the geminal
position of the 7-amino group on the cyclopropylmethylamine
system is effective to reduce the MBI potential. Meanwhile,
compound 2a did not exhibit any inhibitory effects on the
enzymatic activities of other human CYP isoforms over the
range of concentrations tested.16
Table 4 shows effects on human ether-a-go-go-related gene
(hERG) potassium current in hERG transfected CHO-K1 cells.
Compounds that inhibit hERG potassium channels have been
shown to prolong the QT interval of electrocardiogram in man
and increase the risk of fatal cardiac arrhythmia.17 Compound
2a at a dose 30 and 100 μM had no effect on hERG currents.
Comparative studies using other quinolones indicated a
potential of 2a for hERG current that is lower than those of
moxifloxacin and compound 1. The grade of compound 2a was
c
Organism (No. of Strains): LVFX-Intermediate and -Resistant
S. pneumoniae (14)
2a
0.25−2
4−32
8−32
0.25−4
0.06−1
0.12 to >32
0.06 to >32
0.03−4
0.03−8
1
1
1
LVFX
CPFX
MFLX
GRNX
AZM
CAM
AMPC
PCG
8
16
32
4
16
32
4
16
2
0.25
>32
>32
0.06
0.06
0.5
>32
>32
2
1
>32
>32
2
2
2
a
Antibacterial Aactivities were determined using a standard micro-
broth dilution method. Abbreviations are as follows: LVFX,
levofloxacin; CPFX, ciprofloxacin; MFLX, moxifloxacin; GRNX,
garenoxacin; AZM, azythromycin; CAM, clarithromycin; AMPC,
amoxicillin; PCG, benzylpenicillin. MIC50 = MIC at which 50% of
isolates are inhibited. MIC80 = MIC at which 80% of isolates are
inhibited. MIC90 = MIC at which 90% of isolates are inhibited. The
strains were collected by the Levofloxacin Surveillance Group from
patients in Japan in 2004.
b
c
acin, or moxifloxacin) and the other categories of drugs
(azythromycin, clarithromycin, or amoxicillin) that were used
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Table 5. Physicochemical Properties and in Vitro Protein
Binding Ratios of 1, 2a, LVFX, and MFLX
a
b
b
2a
1
LVFX
5.1
MFLX
c
P′
solubility (μg/mL)
protein binding ratio (%)
19.2
620
34.7
8.8
53.8
777
47.8
d
446
41.8
>11900
e
f
32.2
a
The physicochemical parameters (apparent partition coefficient and
aqueous solubility) of 1 and moxifloxacin were assessed by using a free
b
base quinolone compound. Abbreviation are as follows: LVFX,
c
levofloxacin; MFLX, moxifloxacin. Apparent partition coefficient,
d
e
CHCl3/0.1 M phosphate buffer (pH 7.4). Aqueous solubility. In
vitro human plasma protein binding ratios of the quinolones were
measured by an ultrafiltration method at 5 μg/mL compounds. The
data were expressed as the mean values SD of three experiments.
f
The value was examined at 10 μg/mL.
Figure 3. Mechanism-based inhibition against CYP3A4 by 1. Shown
are effects of preincubation of CYP3A4 with 1 for 1′-hydroxylation of
midazolam in pooled human liver microsomal system.
protein binding ratio of 2a was almost the same as the value of
levofloxacin.
The pharmacokinetic profiles of 2a, 1, levofloxacin, and
moxifloxacin after single oral administration to rats are
displayed in Table 6. Compound 2a exhibited higher peak
Table 6. Pharmacokinetic Parameters of 2a and Reference
Quinolones in Rats after an Oral Dose of 5 mg/kg (n = 3)
a
d
PK parameter/route: po
2a
1
LVFX MFLX
serum
Cmax (μg/mL)
1.22
3.08
13.7
4.4
0.816
1.59
6.77
4.3
1.47
3.41
5.57
1.6
1.49
4.46
15.2
3.4
AUC0−8h (μg·h/mL)
AUC0−8h (μg·h/mL)
lung
b
Kp
liver
AUC0−8h (μg·h/mL)
Kp
32.1
10.4
27.8
9.0
15.0
9.4
16.4
4.8
24.5
5.5
b
kidney
AUC0−8h (μg·h/mL)
19.3
12.2
23.1
19.3
5.6
30.3
6.8
b
Kp
c
urinary recovery0−24h (%)
32.0
80.0
25.7
a
b
Animal: 7-week-old male Crj:CD rats. Kp values are tissue/serum
c
concentration ratio. The values were calculated by using the
concentrations of unchanged quinolones. Moxifloxacin hydrochloride
d
Figure 4. Mechanism-based inhibition against CYP3A4 by 2a. Shown
are effects of preincubation of CYP3A4 with 2a for 1′-hydroxylation of
midazolam in pooled human liver microsomal system.
hydrate was administered.
plasma concentration (Cmax) and area under the time−
concentration curve (AUC) than those of 1. Compound 2a
also exhibited a favorable profile of distribution. It was
distributed to the lung, a major target organ, very effectively;
AUC(lung)/AUC(serum) ratio (Kp) of compound 2a was higher
than the value of existing respiratory quinolone, moxifloxacin.
The pharmacokinetic profiles of compound 2a and reference
quinolones after oral or intravenous administration to monkeys
are summarized in Table 7. After single oral administration, the
Table 4. Effects on hERG Potassium Current in hERG
Transfected CHO-K1 Cells
a
% inhibition
compd
30 μM
100 μM
2a
1.9
−0.9
21.5
5.2
2.7
4.2
LVFX
MFLX
1
42.3
14.1
C
max and AUC values of 2a were higher than those of the other
a
Abbreviations are as follows: LVFX, levofloxacin; MFLX, moxi-
quinolones tested. Similarly, 2a administered intravenously
further showed a high AUC value. The bioavailability of
compound 2a was about 70% in monkeys. Compound 2a was
thus expected to exhibit a high plasma concentration and a
good distribution to lung in humans, thereby furnishing
superior efficacy for the respiratory infections. Moreover,
above 60% of an intact 2a was excreted in the urine. Taken
together with the profile of pharmacokinetics and the
antibacterial activity, compound 2a will be expected to show
excellent efficacy against urinary tract infections.
floxacin.
approximately equal to that of levofloxacin, which is known not
to cause QT prolongation syndrome at clinical dosage.18
The apparent partition coefficients (P′),19 the solubility to
water,20 and in vitro plasma protein binding ratios in human of
2a, 1 (as free base substance), and levofloxacin are listed in
Table 5. Introduction of a methyl group into the geminal
position of the 7-amino group on the C7-substituent
significantly increased the P′ of parent compound 1. The P′
of 2a was 2 times higher than that of 1, while the solubility in
water of 2a was slightly improved. On the other hand, the
The results of the intravenous single-dose toxicity study and
the micronucleus test are summarized in Table 8. An
approximate lethal dose in mice was greater than 100 mg/kg.
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Table 7. Pharmacokinetic Parameters of 2a and Reference
a
Quinolones in Monkeys after a Dose of 5 mg/kg (n = 3)
b
PK parameter
2a
1
LVFX
MFLX
Route: po
serum
Cmax (μg/mL)
2.20
16.9
1.27
9.45
3.1
1.92
15.2
3.5
1.03
6.63
5.3
AUC0−24h (μg·h/mL)
t1/2 (h)
4.8
c
urinary recovery0−24h (%)
bioavailability (%)
61.3
41.0
49
73.9
8.1
71
61
route: iv
serum
d
Figure 6. Therapeutic effects on pneumococcal pneumonia (QS-
PRSP) in mice. Animals were CBA/JNCrj mice (n = 4). Bacteria were
S. pneumoniae (QR-PRSP). Test drugs were administered subcuta-
neously twice a day.
C5min (μg/mL)
AUC0−8h (μg·h/mL)
t1/2α (h)
3.66
23.8
4.8
4.75
19.4
2.1
4.17
2.36
10.9
4.8
d
15.5
e
3.2
d
e
urinary recovery0−24h (%)
69.8
50.7
80.7
11.0
a
b
Animal: female cynomolgus monkeys. Moxifloxacin hydrochloride
c
comparable to that of compound 1, although possessing 2-
fold more potency in vitro compared with 2a. The comparison
with GRNX has been previously reported.21
hydrate was administered. The values were calculated by using the
concentrations of unchanged quinolones. These values result from
d
the division of data for 10 mg/kg intravenous administration by
levofloxacin. Those values are the results for a 10 mg/kg intravenous
e
administration.
CONCLUSION
■
Compound 2a, 6-fluoro-8-methoxyquinolone possessing the
(7S)-7-amino-7-methyl-5-azabicyclo[2.4]heptan-5-yl group at
the C-7 position of the scaffold, was designed, synthesized, and
evaluated. Compound 2a exhibited about 4- to 8-fold better
antibacterial activity in vitro against Gram-positive bacteria
compared with the activity of LVFX. Compound 2a also
showed excellent in vivo antibacterial activity against
Streptococcus pneumoniae infection in mice, which was
supported and rationalized by a good pharmacokinetic profile.
In addition, compound 2a did not exert mechanism-based
inhibition on CYP3A4, which was a serious setback of
compound 1.
Compound 2a showed negative responses in the micronucleus
induction at a dose of 150 mg/kg in mice.
Table 8. Intravenous Single-Dose Toxicity and Micronuclei-
Forming Toxicity of 2a in Mice
dose (mg/kg)
50
0/5
100
0/5
150
1/5
intravenous single-dose
a
toxicity/mortality
b
micronuclei-forming toxicity test/result
negative
negative
negative
a
Mortality = (the number of dead mice)/(the number of tested mice).
Using the bone marrow of surviving animals in the intravenous
Thus, compound 2a has been selected for further evaluation
as the promising candidate for treating community-acquired
pneumonia.
b
single-dose toxicity test.
The therapeutic efficacies of compound 2a in experimental
murine pneumonia models due to quinolone-resistant (QR) or
-sensitive (QS) penicillin-resistant Streptococcus pneumoniae
(PRSP) are compared with the efficacies of moxifloxacin and
compound 1 in Figure 5 and Figure 6. The therapeutic efficacy
of subcutaneously administrated 2a was superior to that of
moxifloxacin, while the one in oral administration was
EXPERIMENTAL SECTION
■
General. All melting points were taken on a Yamato MP-500D
melting point apparatus and are uncorrected. Optical rotations were
measured in a 0.5 dm cell at 25 °C at 589 nm with a HORIBA SEPA-
1
300 polarimeter. H NMR spectra were determined on a JEOL JNM-
EX400 spectrometer. Chemical shifts are reported in parts per million
relative to tetramethylsilane or sodium 2,2-dimethyl-2-silapentane-5-
sulfonate as internal standards. Signficant H NMR data are tabulated
1
in the following order: number of protons, multiplicity (s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet), coupling constant(s) in
hertz. High-resolution mass spectra were obtained on a JEOL JMS-700
mass spectrometer under electron impact ionization conditions (EI),
electron spray ionization conditions (ESI), or fast atom bombardment
ionization conditions (FAB). The high-resolution mass spectra were
recorded on a JEOL JMS-100LP spectrometer. Elemental analyses are
indicated only by the symbols of the elements; analytical results were
within 0.4% of the theoretical values. Purities of ≥95% were
determined by elemental analysis (all tested compounds). Column
chromatography refers to flash column chromatography conducted on
Merck silica gel 60, 230−400 mesh ASTM. Thin-layer chromatog-
raphy (TLC) was performed with Merck silica gel 60 F254 TLC
plates, and compound visualization was effected with a 5% solution of
molybdophosphoric acid in ethanol, UV lamp, iodine, or Wako
ninhydrin spray.
Figure 5. Therapeutic effects on pneumococcal pneumonia (QR-
PRSP) in mice. Animals were CBA/JNCrj mice (n = 4). Bacteria were
S. pneumoniae (QR-PRSP). Test drugs were administered subcuta-
neously twice a day.
In Vitro Antibacterial Activity. The MICs of the compounds
tested in this study were determined by the 2-fold microdilution
method using Mueller−Hinton broth (Difco Laboratories, Detroit,
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Journal of Medicinal Chemistry
Article
MI) with an inoculum size of approximately 105 CFU per well. The
MIC was defined as the lowest concentration that prevented visible
bacterial growth after incubation at 35 °C for 18 h.
resolution MS (ESI) calcd for C7H15N2O2: 159.1134. Found:
159.1125.
tert-Butyl (7-Methyl-4-oxo-5-azaspiro[2.4]hept-7-yl)-
carbamate (8). To a solution of 7 (800 mg, 3.46 mmol) in 70 mL
of CH3CN was added 1,1,1,3,3,3-hexamethyldisilazane (7.38 mL, 34.6
mmol) under nitrogen atmosphere, and then the mixture was heated
to reflux for 4 h. After the mixture was cooled at ambient temperature,
MeOH (70 mL) was added. The resultant solution was concentrated
in vacuo. The residue was mixed with Boc2O (1.53 g, 7.00 mmol) and
1,4-dioxane (20 mL), and the mixture was stirred for 5 h at ambient
temperature. Water was added, and the aqueous solution was extracted
with CHCl3 (2×). The combined organic layer was dried over
anhydrous Na2SO4 and concentrated in vacuo. The residue was treated
with light petroleum ether and the solid was collected by filtration to
In Vivo Efficacy against S. pneumoniae Infection in Mice.
Three-week-old male CBA/JNCrlj mice (4 mice/group) were used.
The bacteria used for infection were quinolone-resistant S. pneumoniae
104835 (penicillin-resistant, QR-PRSP) and quinolone-sensitive S.
pneumoniae 033806 (penicillin-resistant, QS-PRSP), both of which
were inoculated by nasal drop. One day after the infection, drugs were
administered subcutaneously or orally twice a day. The number of
bacteria in the lungs was examined on the day after the final
administration of drug. The lungs were removed aseptically and
weighed, and then the viable bacterial counts were determined. The
detection limit was ≥2.30 log10 CFU/g of lung.
Single Intravenous Dose Toxicity. The test compounds were
dissolved in 0.1 N NaOH in saline at different concentrations. The
solution was administered intravenously to male Slc:ddY mice, 6 weeks
of age at a dose level of 50, 100, and 150 mg/kg (10 mL/kg, 0.2 mL/
min). The number of dead mice was counted on day 7.
Bone Marrow Micronucleus Test. The test compounds were
dissolved in 0.1 N NaOH in saline at different concentrations. The
solution was administered intravenously to male Slc:ddY mice, 6 weeks
of age at a dose level of 50, 100, and 150 mg/kg (10 mL/kg, 0.2 mL/
min). At 24 and 48 h after dosing of the compounds, approximately 5
μL of peripheral blood was collected from a tail blood vessel of each
mice. The blood was dropped onto an acridine orange coated glass
slide and covered immediately with a coverslip. For each animal, 1000
reticulocytes were examined for micronuclei by fluorescence
microscopy, and the frequency of micronucleated reticulocytes
(MNRET) was expressed as a percentage. Statistical analysis was
performed by the Kastenbaum and Bowman method.
Pharmacokinetic Studies. Seven-week-old male Crj:CD rats (n =
4) were used. The animals were administered drug samples in a single
intravenous dose (5 mg/kg) as an aqueous solution. The
concentrations of the compounds were determined by a micro-
biological assay (agar well dilution method) using B. subtilis ATCC
6051. The mean values of the four rats were shown.
1
give 8 (502 mg, 60%) as a colorless powder, mp 221−223 °C. H
NMR (CDCl3): δ 0.77−0.82 (1H, m), 0.94−1.04 (2H, m), 1.16−1.23
(1H, m), 1.28 (3H, s), 1.43 (9H, s), 3.29 (1H, d, J = 10.3 Hz), 4.12
(1H, m), 4.60 (1H, br s), 5.82 (1H, br s). High-resolution MS (ESI)
calcd for C12H21N2O3: 241.1552. Found: 241.1538. Anal. Calcd for
C12H20N2O3: C 59.88, H 8.39, N 11.66. Found: C 59.80, H 8.43, N
11.74.
tert-Butyl (5-Benzyl-7-methyl-4-oxo-5-azaspiro[2.4]hept-7-
yl)carbamate (9). To a solution of 8 (3.12 g, 13.0 mmol) in 65
mL of DMF on an ice bath was added NaH (55% mineral oil
dispersion, 566 mg, 13.0 mmol) carefully. After the mixture was stirred
for 40 min at the same temperature, BnBr (1.85 mL, 15.6 mmol) was
added dropwise and the mixture was stirred for 1.5 h at ambient
temperature. The reaction mixture was diluted with 300 mL of AcOEt
and washed with water (2×) and brine. The organic solution was dried
over anhydrous Na2SO4 and concentrated in vacuo. The resultant
residue was purified by silica gel column chromatography, eluting with
1
hexane/EtOAc = 4:1 to yield 9 (4.20 g, 98%) as a colorless oil. H
NMR (CDCl3): δ 0.76−0.81 (1H, m), 0.93−1.06 (2H, m), 1.21−1.29
(4H, m), 1.37 (9H, s), 3.14 (1H, d, J = 10.3 Hz), 3.92−3.98 (1H, m),
4.44, 4.56 (each 1H, ABq, J = 14.9 Hz), 4.56 (1H, br s), 7.22−7.33
(5H, m). High-resolution MS (ESI) calcd for C19H27N2O3: 331.2022.
Found: 331.2020.
(−)-tert-Butyl (5-Benzyl-7-methyl-4-oxo-5-azaspiro[2.4]-
hept-7-yl)carbamate ((−)-9) and (+)-tert-Butyl (5-Benzyl-7-
methyl-4-oxo-5-azaspiro[2.4]hept-7-yl)carbamate ((+)-9). An
amount of 2.25 g of racemic 9 was separated into its enantiomers
by semipreparative HPLC by using a Chiralpak AD column (Daicel
Chemical Industries, Ltd.; 250 mm × 20 mm, 5 μm; flow, 20 mL/min;
solvents, hexane/isopropanol 90:10; 50 mg/run; UV detection at 254
nm) to give (−)-9 (997 mg, 44%, tR = 7.0 min) and (+)-9 (957 mg,
43%, tR = 11.3 min) as a colorless powder, respectively. (−)-9: mp
109−111 °C. [α]2D5 −113.9° (c 0.180, CHCl3). Anal. Calcd for
C19H26N2O3: C 69.09, H 7.93, N 8.48. Found: C 68.80, H 7.99, N
8.51. (+)-9; mp 108−110 °C. [α]2D5 +108.8° (c 0.249, CHCl3). Anal.
Calcd for C19H26N2O3: C 69.09, H 7.93, N 8.48. Found: C 68.99, H
8.01, N, 8.48.
(−)-tert-Butyl (5-Benzyl-7-methyl-5-azaspiro[2.4]hept-7-yl)-
carbamate ((−)-10). To a solution of (−)-9 (950 mg, 2.88 mmol)
in CH2Cl2 (15 mL) was added trifluoroacetic acid (7.5 mL) at ambient
temperature. The mixture was stirred for 40 min and then
concentrated in vacuo. The resultant residue was mixed with toluene
and concentrated in vacuo (2×). After saturated aqueous NaHCO3
was added, the aqueous layer was extracted with CHCl3 (2×). The
combined organic solution was dried over anhydrous Na2SO4 and
concentrated in vacuo. To the solution of the residue in THF (30 mL)
was added LiAlH4 (218 mg, 5.75 mmol) at 0 °C, and the mixture was
stirred for 1 h at same temperature. After addition of LiAlH4 (109 mg,
2.87 mmol), the mixture was stirred for 3 h at ambient temperature
and stirred at 0 °C again. To the mixture, water (0.31 mL), 15 wt %
aqueous NaOH (0.31 mL), and water (0.93 mL) were added
dropwise. The resultant mixture was stirred at ambient temperature for
14 h and dried over anhydrous MgSO4. The suspension was filtered
through Celite, and the filtrate was concentrated in vacuo. The residue
was mixed with Boc2O (1.26 g, 5.75 mmol) and CH2Cl2 (15 mL), and
the mixture was stirred for 20 h at ambient temperature and then
X-ray Crystallographic Analysis of 14. A colorless prism-shaped
crystal was formed from AcOEt: C20H27NO3; FW = 329.44; sample
dimensions, 0.40 mm × 0.30 mm × 0.34 mm. The lattice parameters
and intensities were measured on a Rigaku AFC7R diffractometer (Cu
Kα radiation, λ = 1.541 78 Å, graphite monochromator, ω−2θ scans,
2θmax = 120.1°): orthorhombic, space group P212121; a = 16.396(2) Å,
b = 17.122(2) Å, c = 6.800(1) Å, V = 1908.9(5) Å3, Z = 4; Dcalcd = 1.15
g/cm3; F000 = 712; μ = 6.09 cm−1. The structure was solved by direct
methods with the program Sir92.22 The final cycle of full-matrix least-
squares refinement was based on 1665 observed reflections and 245
variable parameters and converged at R = 0.063 (Rw = 0.191).
1-(1,2-Diamino-1-methylethyl)-1-cyclopropanecarboxylic
Acid Dihydrochloride (7). To a solution of 5 (9.21 g, 50.0 mmol) in
7 N NH3−MeOH (300 mL) were added aqueous NH3 (28%, 90 mL),
ammonium chloride (53.5 g, 1.00 mol), and sodium cyanide (4.90 g,
100 mmol) at 0 °C, and the mixture was stirred at ambient
temperature for 18 h. The mixture was concentrated in vacuo. Water
(100 mL) was added to the residue, and then the aqueous solution was
extracted with CH2Cl2 (3×). The organic layers were combined, dried
over anhydrous Na2SO4, and concentrated to give 6 (10.15 g) as a pale
brown oil. To a solution of 6 (1.12 g, 5.30 mmol) in EtOH (50 mL)
was added Raney nickel catalyst (about 50%, 10 mL), and the mixture
was stirred at ambient temperature for 6 h under hydrogen
atmosphere. The resultant suspension was filtered through Celite,
and the filtrate was concentrated in vacuo. The residue was dissolved
in concentrated aqueous HCl (5 mL), and the mixture was stirred at
ambient temperature for 0.5 h. After the mixture was diluted with
water, the resultant solution was concentrated in vacuo. The resultant
residue was mixed with EtOH and concentrated in vacuo (2×) to give
7 (0.82 g, 65%) as a yellow amorphous product. 1H NMR (CD3OD):
δ 1.20−1.26 (1H, m), 1.28 (3H, s), 1.32−1.43 (2H, m), 1.58−1.62
(1H, m), 3.46 (1H, d, J = 13.4 Hz), 3.80 (1H, d, J = 13.4 Hz). High-
1980
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Journal of Medicinal Chemistry
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concentrated in vacuo. The residue was purified by silica gel column
chromatography, eluting with CHCl3/MeOH/triethylamine = 95:5:1
to yield (−)-10 (586 mg, 64%) as a colorless oil. 1H NMR (CDCl3): δ
0.40−0.45 (1H, m), 0.50−0.55 (1H, m), 0.63−0.69 (1H, m), 0.80−
0.85 (1H, m), 1.20 (3H, s), 1.43 (9H, s), 2.44 (1H, d, J = 8.8 Hz), 2.59
(1H, d, J = 9.5 Hz), 2.83 (1H, d, J = 8.8 Hz), 3.33 (1H, m), 3.57 (1H,
d, J = 13.2 Hz), 3.68 (1H, d, J = 13.2 Hz), 4.75 (1H, br s), 7.20−7.37
(5H, m). High-resolution MS (ESI) calcd for C19H29N2O2: 317.2229.
Found: 317.2253. [α]2D5 −63.6° (c 0.129, CHCl3).
residue in THF (450 mL) on an ice bath was added 1 M TBAF/THF
(148 mL) dropwise, and the mixture was stirred for 2 h at ambient
temperature. After concentration in vacuo, AcOEt and 10% aqueous
NaHCO3 were added to the mixture. The organic solution was washed
with 10% aqueous citric acid and brine, dried over anhydrous Na2SO4,
and concentrated in vacuo. The resulting residue was purified by silica
gel column chromatography, eluting with hexane/AcOEt = 1:1 to yield
diastereomixture 13 (29.1 g, 85%) as a colorless oil. 1H NMR
(CDCl3): δ 1.28 (3H, s), 1.40 (9H, s), 1.51−1.53 (1H, m), 1.53 (3H,
d, J = 7.1 Hz), 1.78−1.94 (2H, m), 2.90−3.08 (2H, m), 3.67−3.75
(1H, m), 3.80−3.91 (1H, m), 4.85−4.89 (1H, m), 5.43−5.53 (1H, m),
7.27−7.37 (5H, m). High-resolution MS (ESI) calcd for C20H30NO4:
348.2175. Found: 348.2177.
tert-Butyl (7S)-7-Methyl-4-oxo-5-[(R)-1-phenylethyl]-5-
azaspiro[2.4]heptan-7-carboxylate (14). To a solution of 13
(29.1 g, 83.9 mmol) in CH2Cl2 (280 mL) on an ice bath were added
TEA (15.2 mL, 109 mmol), benzenesulfonyl chloride (11.8 mL, 92.3
mmol), and DMAP (1.02 g, 8.39 mmol). The mixture was stirred at
ambient temperature for 19 h and diluted with AcOEt. This solution
was washed with saturated aqueous NH4Cl, 1 N aqueous HCl,
saturated aqueous NaHCO3, and brine. The organic layer was dried
over anhydrous Na2SO4 and concentrated in vacuo. To a solution of
the residue in anhydrous THF (470 mL) on an ice bath was added 1
M NaHMDS/THF (109 mL) under nitrogen atmosphere, and the
mixture was stirred at ambient temperature for 1 h. Aqueous saturated
NH4Cl was added to the mixture, and the resultant solution was
extracted with AcOEt (2×). The organic layer was washed with brine,
dried over anhydrous Na2SO4, and concentrated in vacuo. The
resultant residue was purified by silica gel column chromatography,
eluting with hexane/AcOEt = 3:1 to yield 14 (24.6 g, 89%) as a
colorless crystal, mp 55−57 °C. 1H NMR (CDCl3): δ 0.72−0.77 (1H,
m), 0.85−0.90 (1H, m), 1.04−1.13 (2H, m), 1.18 (3H, s), 1.32 (9H,
s), 1.54 (3H, d, J = 7.1 Hz), 3.08 (1H, d, J = 9.8 Hz), 3.53 (1H, d, J =
7.1 Hz), 5.52 (1H, q, J = 7.1 Hz), 7.26−7.34 (5H, m). High-resolution
MS (FAB) calcd for C20H28NO3: 330.2069. Found: 330.2069. [α]D25
+122.1° (c 0.517, CHCl3). Anal. Calcd for C20H27NO3: C 72.92, H
8.26, N 4.25. Found: C 72.64, H 8.27, N 4.06.
7-(7-Amino-7-methyl-5-azaspiro[2.4]hept-5-yl)-6-fluoro-1-
[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-4-oxo-1,4-dihydro-
quinoline-3-carboxylic Acid (2a). To a solution of (−)-10 (581
mg, 1.84 mmol) in MeOH (40 mL) was added 10% Pd/C (M, 290
mg, containing 50% water), and the mixture was stirred for 5 h at
ambient temperature under hydrogen atmosphere. The catalyst was
filtered off, and the filtrate was concentrated in vacuo. The residue was
mixed with 4 (663 mg, 1.84 mmol), triethylamine (0.795 mL, 5.52
mmol), and DMSO (5 mL), and the mixture was stirred at 40 °C for
14 h. To this solution were added 80% aqueous EtOH (50 mL) and
triethylamine (5 mL), and the resultant mixture was heated to reflux
for 2 h and then concentrated in vacuo to give the residue, which was
diluted with AcOEt. The organic solution was washed with 10%
aqueous citric acid solution, water (2×) and brine. The organic layer
was dried over anhydrous Na2SO4 and concentrated in vacuo. To the
residue was added 10 mL of concentrated aqueous HCl at 0 °C, and
the mixture was stirred for 20 min at ambient temperature. The
aqueous solution was washed with CHCl3 and made alkaline with
saturated aqueous NaOH at 0 °C. The pH of the solution was adjusted
to 7.4 with concentrated aqueous HCl and then diluted aqueous HCl.
The resultant solution was extracted with CHCl3 (2×) and CHCl3/
MeOH = 9:1. The combined organic solution was dried over
anhydrous Na2SO4 and concentrated in vacuo. The crude product was
recrystallized from EtOH to yield 2a (644 mg, 84%) as a pale pinkish
1
powder, mp 198−200 °C. H NMR (0.1 N NaOD/D2O): δ 0.49−
0.56 (2H, m), 0.67−0.76 (2H, m), 1.12 (3H, s), 1.43−1.64 (2H, m),
3.56 (3H, s), 3.59−3.71 (4H, m), 3.99−4.04 (1H, m), 4.80−5.03 (1H,
m), 7.65 (1H, d, J = 13.9 Hz), 8.45 (1H, s). [α]2D5 +40.8° (c 0.147, 0.1
(7S)-7-(tert-Butoxycarbonylamino)-7-methyl-5-[(R)-1-phe-
nylethyl]-5-azaspiro[2.4]heptane (16). To a solution of 14 (24.5
g, 74.4 mmol) in CH2Cl2 (120 mL) on an ice bath was added
trifluoroacetic acid (120 mL), and the mixture was stirred at ambient
temperature for 2 h and concentrated in vacuo. The residue was mixed
with toluene, and the solution was concentrated in vacuo. After the
residue was dissolved in 1 N aqueous NaOH at 0 °C, the aqueous
solution was washed with AcOEt and acidified with concentrated
aqueous HCl under ice-cooling. The mixture was extracted with
CHCl3 (2×), and the combined organic solution was washed with
water and brine, dried over anhydrous Na2SO4, and concentrated in
vacuo. To a solution of the residue in toluene (200 mL) were added
triethylamine (20.7 mL, 149 mmol) and diphenylphosphoryl azide
(17.6 mL, 81.8 mmol) at ambient temperature. The mixture was
heated to reflux for 1 h and then concentrated in vacuo. The residue
was mixed with 1,4-dioxane (180 mL), water (90 mL), and
concentrated aqueous HCl (90 mL), and the mixture was stirred at
50 °C for 1 h. Water (200 mL) was added to the mixture, and the
resultant solution was washed with AcOEt and made alkaline with 10
N aqueous NaOH at 0 °C and extracted with toluene (2×). The
combined organic solution was washed with brine and dried over
anhydrous Na2SO4 and concentrated in vacuo. To a solution of the
residue in toluene (82 mL) was added Red-Al (65% w/w in toluene,
77.6 mL, 259 mmol) dropwise over 15 min, and the mixture was
stirred at 80 °C for 10 min. After the mixture was cooled to 0 °C, 25
wt % aqueous NaOH (158 mL) was dropped into the mixture, which
was extracted with toluene. To the organic solution which was washed
with brine was added Boc2O (15.6 g, 71.2 mmol), and the mixture was
stirred at ambient temperature for 3 h and concentrated in vacuo. The
residue was purified by silica gel column chromatography, eluting with
N
a q u e o u s N a O H ) . A n a l . C a l c d f o r
C21H23F2N3O4·0.75EtOH·0.5H2O: C 58.37, H 6.20, F 8.20, N 9.08.
Found: C 58.23, H 5.99, F 8.09, N 9.02.
(+)-tert-Butyl (5-Benzyl-7-methyl-5-azaspiro[2.4]hept-7-yl)-
carbamate ((+)-10). With the procedures as described for (−)-10,
the title compound was prepared in 69% yield from (+)-9 as a
1
colorless oil. H NMR (CDCl3): δ 0.40−0.45 (1H, m), 0.50−0.55
(1H, m), 0.63−0.69 (1H, m), 0.80−0.85 (1H, m), 1.20 (3H, s), 1.43
(9H, s), 2.44 (1H, d, J = 8.8 Hz), 2.59 (1H, d, J = 9.5 Hz), 2.83 (1H, d,
J = 8.8 Hz), 3.33 (1H, m), 3.57 (1H, d, J = 13.2 Hz), 3.68 (1H, d, J =
13.2 Hz), 4.75 (1H, br s), 7.20−7.37 (5H, m). High-resolution MS
(ESI) calcd for C19H29N2O2: 317.2229. Found: 317.2249. [α]2D5 +76.2°
(c 0.290, CHCl3).
7-(7-Amino-7-methyl-5-azaspiro[2.4]hept-5-yl)-6-fluoro-1-
[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-4-oxo-1,4-dihydro-
quinoline-3-carboxylic Acid (2b). With the procedures as described
for 2a, the title compound was prepared in 78% yield from (+)-10 as a
pale pinkish powder, mp 212−214 °C. 1H NMR (0.1 N NaOD/D2O):
δ 0.52 (2H, m), 0.73 (2H, m), 1.07 (3H, s), 1.42−1.64 (2H, m), 3.45
(1H, d, J = 10.3 Hz), 3.52−3.56 (1H, m), 3.55 (3H, s), 3.73 (1H, dd, J
= 2.2, 10.0 Hz), 3.85 (1H, d, J = 9.0 Hz), 3.99−4.04 (1H, m), 4.82−
5.02 (1H, m), 7.64 (1H, d, J = 14.4 Hz), 8.45 (1H, s). [α]2D5 +128.8° (c
0 . 1 6 3 , 0 . 1
N a q u e o u s N a O H ) . A n a l . C a l c d f o r
C21H23F2N3O4·1.0EtOH·0.5H2O: C 58.22, H 6.37, F 8.01, N 8.86.
Found: C 58.02, H 6.13, F 8.05, N 9.02.
tert-Butyl (3S)-4-(2-Hydroxyethyl)-3-methyl-5-oxo-1-[(R)-1-
phenylethyl]pyrrolidine-3-carboxylate (13). Under nitrogen
atmosphere, to a solution of 11 (30.0 g, 98.9 mmol) and 12 (36.8
g, 129 mmol) in anhydrous THF (288 mL) was added 1 M LiHMDS/
THF (129 mL) dropwise at −4 °C. After the mixture was stirred at 2
°C for 3.5 h, saturated aqueous NH4Cl and AcOEt were added. The
separated organic solution was washed with brine, dried over
anhydrous Na2SO4, and concentrated in vacuo. To a solution of the
1
hexane/AcOEt = 3:1 to yield 16 (18.0 g, 73%) as a colorless oil. H
NMR (CDCl3): δ 0.37−0.49 (2H, m), 0.62−0.68 (1H, m), 0.77−0.82
(1H, m), 1.20 (3H, s), 1.32 (3H, d, J = 6.6 Hz), 1.44 (9H, s), 2.46
1981
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Journal of Medicinal Chemistry
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(2H, dd, J = 33.2, 9.3 Hz), 2.68 (1H, d, J = 8.8 Hz), 3.27 (1H, q, J =
6.6 Hz), 3.31−3.34 (1H, m), 4.71 (1H, s), 7.19−7.34 (5H, m). High-
resolution MS (ESI) calcd for C20H31N2O2: 331.2385. Found:
331.2406. [α]2D5 −23.817° (c 0.786, CHCl3).
treatment of resistant pneumococcal infections. Expert Opin. Invest.
Drugs 2005, 14 (8), 973−995.
(6) Poluzzi, E.; Raschi, E.; Motola, D.; Moretti, U.; De Ponti, F.
Antimicrobials and the risk of torsades de pointes: the contribution
from data mining of the US FDA Adverse Event Reporting System.
Drug Saf. 2010, 33 (4), 303−314. Falagas, M. E.; Rafailidis, P. I.;
Rosmarakis, E. S. Arrhythmias associated with fluoroquinolone
therapy. Int. J. Antimicrob. Agents 2007, 29 (4), 374−379. Owens, R.
C., Jr.; Ambrose, P. G. Torsades de pointes associated with
fluoroquinolones. Pharmacotherapy 2002, 22 (5), 663−668.
(7) Mehlhorn, A. J.; Brown, D. A. Safety concerns with
fluoroquinolones. Ann Pharmacother. 2007, 41 (11), 1859−1866.
Sprandel, K. A.; Rodvold, K. A. Safety and tolerability of
fluoroquinolones. Clin Cornerstone 2003, No. Suppl. 3, S29−S36.
(8) Kawakami, K.; Ohki, H.; Kimura, K.; Tanaka, M.; Takemura, M.;
Sato, K.; Takahashi, H.; Hayakawa, I. DK-507k, a New 8-
Methoxyquinolones: Synthesis and Biological Evaluation of 7-[(3-
Amino-4-substituted)pyrrolidine-1-yl] Derivatives. Abstracts of Papers,
41st Interscience Conference on Antimicrobial Agents and Chemo-
therapy, 2001; American Society for Microbiology: Washington, DC;
Abstract F-546.
7-[(7S)-7-Amino-7-methyl-5-azaspiro[2.4]hept-5-yl]-6-fluo-
ro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-4-oxo-1,4-dihy-
droquinoline-3-carboxylic Acid (2). To a solution of 16 (18.0 g,
54.5 mmol) in MeOH (180 mL) was added 10% Pd/C (M, 9.0 g,
containing 50% water), and the mixture was stirred at ambient
temperature for 18 h and then at 40 °C for 5.5 h under hydrogen
atmosphere. The catalyst was filtered off, and the filtrate was
concentrated in vacuo, followed by the procedures as described for
2a. The title compound was prepared in 79% yield from 16 as a pale
pinkish powder. The analytical data of this compound corresponded
with those of compound 2a.
ASSOCIATED CONTENT
* Supporting Information
Crystallographic details for 14. This material is available free of
■
S
charge via the Internet at http://pubs.acs.org.
(9) Dresser, G. K.; Spence, J. D.; Bailey, D. G. Pharmacokinetic-
pharmacodynamic consequences and clinical relevance of cytochrome
P450 #A4 inhibition. Clin. Pharmacokinet. 2000, 38, 41−57. Kalgutkar,
A. S.; Obach, R. S.; Maurer, T. S. Mechanism-based inactivation of
cytochrome P450 enzymes: chemical mechanisms, structure−activity
relationships and relationship to clinical drug−drug interactions and
idiosyncratic adverse drug reactions. Curr Drug Metab. 2007, 8 (5),
407−447.
AUTHOR INFORMATION
Corresponding Author
*Phone: +81-3-3492-3131. Fax; +81-3-5436-8563. E-mail:
odagiri.takashi.fb@daiichisankyo.co.jp.
■
Notes
The authors declare no competing financial interest.
(10) Thompson, R. A.; Isin, E. M.; Li, Y.; Weaver, R.; Weidolf, L.;
Wilson, I.; Claesson, A.; Page, K.; Dolgos, H.; Kenna, J. G. Risk
assessment and mitigation strategies for reactive metabolites in drug
discovery and development. Chem.-Biol. Interact. 2011, 192, 65−71.
Omar, M. A.; Wilson, J. P. FDA adverse event reports on strain-
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Utrehit, J. Idiosyncratic drug reactions: past, present, and future. Chem.
Res. Toxicol. 2008, 21 (1), 84−92.
ACKNOWLEDGMENTS
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We thank Dr. Kiyoshi Takasuna and Koichi Goto for the safety
assessment. And we thank Dr. Masaya Tachibana for the MBI
assay. In particular, we thank Megumi Chiba, Dr. Ryo
Okumura, and Dr. Yuichi Kurosaka for the biological tests.
We also thank Hidetaka Sakurai for the X-ray crystal analyses.
(11) Silverman, R. B.; Ding, C. Z.; Borrillo, J. L.; Chang, J. T.
Mechanism-based enzyme inactivation via a diactivated cyclopropane
intermediate. J. Am. Chem. Soc. 1993, 115, 2982−2983.
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